FRET probes for analysis of serpin-mediated apoptosis in focal brain ischemia. In light of the profound significance of apoptosis research, it s essential to have probes which can discriminate between different apoptotic pathways. The serine protease- dependent pathway is one of the most recently identified apoptotic programs, which still lacks specific tools for its visualization in situ. Serpin 1B is a key member of this pathway and is an important effector of apoptotic death in neural tissue. In metabolic stress it transforms into an active endonuclease. This unique transition occurs in hypoxia and simultaneously exposes an active DNase site and a nuclear localization signal. The newly formed DNA degrading enzyme promptly moves into the nucleus causing cell death. In spite of the high importance of this non-caspase apoptosis mechanism, there are currently no methods to selectively label it in tissue sections and in live cell cultures. In this project we will develp such methods and will apply them to study serpin-mediated apoptosis in focal brain ischemia. Specific aims: 1. To develop the new FRET-based approach for selective labeling of serpin-mediated apoptosis in tissue sections. The approach will simultaneously co-detect serpin 1B-derived endonuclease protein and characteristic DNA breaks which it produces. 2. To develop the new FRET-based approach for selective labeling of serpin-mediated apoptosis in live cell cultures. The FRET probes will become fluorescent only after they detect a specific marker of serpin-mediated pathway. 3. To apply the newly developed molecular tools to study focal cerebral ischemia. To evaluate the role of serpin-mediated apoptosis in focal brain ischemia in the rat model; to investigate its initiation mechanisms, dynamics, and patterns of distribution. The project will introduce enabling technologies for apoptosis research in general, and for studies in ischemia in particular. Their first systematic application to focal brain ischemia will provide information useful for the future clinical and research investigations of stroke, and for the development of effective therapeutic interventions.